Evaluation of cathepsin B levels in fresh thighs selected for cured raw ham production (original) (raw)
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Assay of Cathepsin D activity in fresh pork muscle and dry-cured ham
Meat Science, 1991
A BS TRA C T Different assays of cathepsin D activity in both porcine muscle and dry-cured ham extracts have been tested in order to find the best conditions for a reliable detection of cathepsin D in dry-cured meat products. The enzyme was effectively extracted with 0"2% (v/v) of Triton X-IO0. Nucleases if present were not observed to interfere with the assays. The best conditions for a reliable detection of cathepsin D in dry-cured ham were found to be the incubation of the enzyme extract for 1 h at 45°C in a reaction mixture containing 0"60% (w/v) of haemoglobin in 0"2 M sodium citrate buffer, pH = 3"7.
Problems associated with the assay of cathepsin D in meat and meat products
Food Chemistry, 1991
Cathepsin D is usually assayed by following the release of the trichloroacetic (TCA)-soluble peptides from denatured haemoglobin at 280nm, but some artefacts may appear giving false results. Cathepsin D activity has therefore been assayed under different conditions in muscle, liver and dry-cured ham extracts. Substantial errors (around 50-56 %) become evident when using the classical standard assay. The assay of cathepsin D activity in muscle extracts should include the use of a blank containing a specific inhibitor such as isovaler ylpepstatin.
Relationship between raw ham cathepsin B activity and firmness of dry cured hams
Italian Journal of Animal Science, 2010
This study aimed to investigate the relationship between cathepsin B activity and muscle firmness of dry cured hams. A total of 988 samples of semimembranosus muscle were collected from raw hams of heavy pigs and cathepsin B activity was determined using fluorimetric method. Raw hams were cured following San Daniele guidelines. Dry-cured hams were deboned and cross-sectioned. On the cross section firmness was measured at three muscular sites (M. semimembranosus, semitendinosus and biceps femoris) using a Hardness Meter MK2. This study did not evidence any significant relationship between cathepsin activity and firmness of dry cured hams.
The genetic relationship between enzymatic activity of cathepsin B and firmness of dry-cured hams
Meat Science, 2008
This study aimed to investigate the genetic relationship between enzymatic activity of raw hams and firmness of dry-cured hams. Instrumental firmness and sensory panel firmness scores were obtained from 2058 and 3275 dry-cured hams, respectively. On a sub-sample of 988 raw hams for dry-curing, enzymatic activity of cathepsin B was determined through a fluorimetric analytical method. A multiple-trait animal model was used to estimate heritability and genetic correlations of these traits. Estimates of heritability were moderate, ranging from 0.11 for sensory panel firmness scores to 0.25 for cathepsin B activity. Genetic correlations between firmness measures on dry-cured hams and enzymatic activity of raw hams were low. The efficiency of a selection program aimed to reduce the incidence of excessive softness in dry-cured hams on the basis of enzymatic activity of cathepsin B of raw hams is expected to be very limited.
Meat science, 1999
Pork muscle cathepsins (B, B+L, and H), cysteine proteinase inhibitors and lipolytic enzyme activities were measured in the ospring of ®ve dierent genetic sire types: Danish Duroc (DU), Dutch Large White (LW D), English Large White (LW E), Belgian Landrace  Landrace (BLÂLR) and Belgian Landrace (BL). Cathepsin B and B+L activities were higher for LW E and LW D sires than for BLÂLR and BL. Cathepsin H activity showed an opposite evolution, being higher for BL and BLÂLR sires than for DU, LW D and LW E. Cysteine proteinase inhibitor activity was higher for LW E sires than for DU and BL. In lipolytic enzymes, BL sires had a lower acid lipase activity than DU and LW E sires and also a lower neutral esterase activity than LW E and LW D sires. Sig-ni®cant dierences between sexes were found for cathepsin H activity only, being higher for females.
The use of muscle enzymes as predictors of pork meat quality
Food Chemistry, 2000
Muscle endo-protease (calpains and cathepsins) and exo-protease (dipeptidyl-peptidases and aminopeptidases) activities were assayed at 2 h post-mortem in different meat qualities (PSE, RSE, RFN and DFD). The sensory characteristics of the different pork meat qualities were also evaluated in order to correlate them to the proteolytic activity. The assay of aminopeptidase and dipeptidylpeptidase activities (AAP, RAP, LAP, DPPI and DPPIV) at 2 h post-mortem discriminate between exudative and non-exudative classes explaining 74.6% of the variability. Also, at 24 h post-mortem 71.2% of the variability was detected by the measurement of PGAP, AAP, RAP, DPPII and DPPIV. Therefore, the exoprotease activities can constitute a novel and adequate technique to predict early post-mortem pork meat quality allowing its assay till 24 h post-mortem because of the good stability of the enzymes during this post-mortem time.
Catalytic and physico-chemical characteristics of goat spleen cathepsin B
IUBMB Life, 1997
To improve the level of purity of cathepsin B, we have modified the published procedure [Agarwal, S.K. and Khan, M.Y. (1987) Biochem. Int. 15, 785-792] by incorporating CM-Sephadex ion exchange chromatography and chromatofocusing. The enzyme thus isolated could be resolved into one 26 kDa major and a minor 27 kDa protein bands on SDS-PAGE. The two components, however, could not be separated by gel filtration and they eluted, in a single peak corresponding to a molecular mass of 28.1 kDa . Among the various substrates tested, Z-Phe-Arg-MCA with a K of 0.058 mM and hemoglobin with a K of 1.449 ~tM were the most m preferred synthetic and protein substrates respectively. It was found to be a glycoprotein with an acidic pI of 4.8. The enzyme was activated by various thiol-reducing reagents and inhibited by cysteine proteinase inhibitors, divalent cations, lysyl group modifiers, a_nti-inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. !mmunodiffusion studies with anti-goat cathepsin B indicated a tissue/ species dependence.
Animal Genetics, 2002
Excessive softness is a serious defect of dry cured hams which seems related to high activity of lysosomal cysteine proteinases, such as cathepsin B, in fresh pork muscles a few days after slaughtering. As it has been shown that cathepsin B activity has a moderate heritability in Italian Large White pigs we started a candidate gene approach to identify the gene(s) that affect(s) this parameter. Here, we studied two candidate genes: cathepsin B (CTSB) and cystatin B (CSTB). We amplified and sequenced porcine DNA fragments for these two genes that were used to identify polymorphisms by SSCP and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Four and two alleles were detected at the CTSB and CSTB loci, respectively. Sequencing of the CSTB alleles showed a missense mutation that changes a codon for aspartic acid into a codon for asparagine in exon 3 of the gene. Allele frequencies for the two loci differed among the pig breeds studied (Large White,
Studies on activation and inhibition of cathepsin B from buffalo liver
Journal of Protein Chemistry, 1996
Cathepsin B (EC3.4.22.1) was purified from buffalo liver. The enzyme activity against a-benzoyl-DL-arginine-naphthylamine (BANA) was substantially reduced by heat (above 37~ and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-l-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu 2+ (20-200/zM) and Ca 2+ (30-250mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), and p-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity.